Experimental Hematology 2016;44:352–357

Leukemia and chromosomal instability in aged Fancc�/� mice

Donna Cerabonaa,b, Zejin Suna, and Grzegorz Nalepaa,b,c,d

aDepartment of Pediatrics, Indiana University School of Medicine, Indianapolis, IN; bDepartment of Biochemistry and Molecular Biology, Indiana

University School of Medicine, Indianapolis, IN; cDepartment of Medical and Molecular Genetics, Indiana University School of Medicine,

Indianapolis, IN; dDivision of Pediatric Hematology–Oncology, Riley Hospital for Children, Indianapolis, IN

(Received 14 January 2016; accepted 29 January 2016)

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Fanconi anemia (FA) is an inherited disorder of genomic instability associated with high riskof myelodysplasia and acute myeloid leukemia (AML). Young mice deficient in FA core com-plex genes do not naturally develop cancer, hampering preclinical studies on malignant hema-topoiesis in FA. Here we describe that aging Fancc�/� mice are prone to genomically unstableAML and other hematologic neoplasms. We report that aneuploidy precedes malignant trans-formation during Fancc�/� hematopoiesis. Our observations reveal that Fancc�/� micedevelop hematopoietic chromosomal instability followed by leukemia in an age-dependentmanner, recapitulating the clinical phenotype of human FA and providing a proof of conceptfor future development of preclinical models of FA-associated leukemogenesis. Copyright �2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

The Fanconi anemia (FA) signaling network protectsgenomic integrity and prevents cancer by facilitating inter-phase DNA repair and orchestrating cell division [1–3].Germline biallelic mutations of any FA genes cause Fan-coni anemia, an inherited bone marrow failure syndromeassociated with myelodysplasia (MDS) and acute myeloidleukemia (AML). The overall risk of leukemia in FA isincreased six hundredfold [4].

Young mice deficient in core FA genes do not spontane-ously recapitulate clinical hematopoietic manifestations ofFanconi anemia [5]. Fancc�/� mice demonstrate hypersen-sitivity to cross-linking agents [6], decreased hematopoieticstem cell repopulating ability [7,8], and hypersensitivity tointerferon-g [8], reflecting disruption of the FA signalingnetwork during hematopoiesis. However, young Fancc�/�

mice do not develop spontaneous leukemia or bone marrowfailure [6,8]. One observation study of a small Fancc�/�

mouse cohort (n 5 8) did not detect decreased survival[9]. However, FA patients rarely develop AML in their firstyear of life [10], and two soft tissue tumors (adenocarci-noma and histiocytic sarcoma) have been reported inO13-month-old Fancc�/� mice [11]. Thus, we hypothe-sized that aging Fancc�/� mice may be predisposed to

to: Grzegorz Nalepa, Wells Center for Pediatric

niversity School of Medicine, 1044 West Walnut

apolis, IN 46202; E-mail: gnalepa@iu.edu

� 2016 ISEH - International Society for Experimental Hem

se (http://creativecommons.org/licenses/by-nc-nd/4.0/).

.1016/j.exphem.2016.01.009

hematopoietic malignancies. If the absolute time to theonset of leukemia is similar in FA humans and mice,long-term observation of FA�/� mice may be crucial todetection of cancer predisposition. To address this transla-tionally relevant question, we asked whether Fancc�/�

mice develop malignancies as they age.

Methods

MiceC57Bl/6J Fancc�/� mice were a gift of David W. Clapp (IndianaUniversity). Mice were genotyped by polymerase chain reaction(PCR), as described [9]. B6.SJL-PtprcaPepcb/BoyJ mice were pur-chased from the Indiana University In Vivo Therapeutics Core. Allstudies were approved by the Institutional Animal Care and UseCommittee at Indiana University.

Marrow harvest and transplantationBone marrow cells were flushed from mouse femurs using a 23-gauge needle/syringe (Becton Dickinson). Light-density mononu-clear cells (LDMNCs) were isolated by density gradient usingHistopaque-1119 (Sigma) and centrifuging for 30 min at1,800 rpm with no brake. After centrifugation, LDMNCs wereremoved from the interface and used for experiments. Cytospinswere made by resuspending LDMNCs in phosphate-buffered sa-line (PBS) and centrifuging onto slides at 450 rpm for 5 min ona Shandon Cytospin 3 Cytocentrifuge (Thermo Scientific). Donortest LDMNCs (1.5 � 106, C57Bl/6J background) and donor

atology. Published by Elsevier Inc. This is an open access article under the

353D. Cerabona et al./ Experimental Hematology 2016;44:352–357

competitor BoyJ LDMNCs (1.5 � 106) were transplanted into re-cipients via tail vein injection. Recipients were 8-week-old femaleB6.SJL-PtprcaPepcb/BoyJ mice that underwent whole-body split-dose 1,100-rad irradiation (700 rads/400 rads, 4 hours apart).For chimerism analysis, peripheral blood was collected fromlateral tail veins into EDTA-coated tubes, incubated with redblood cell lysis solution (Qiagen) for 10 min at room temperature,washed, stained with anti-Cd45.2-flluorescein isothiocyanate (BDBiosciences) and anti-Cd45.1-phycoerythrin (BD Biosciences), asdescribed [9], and analyzed on a FacsCalibur machine (Becton-Dickinson). At least 10,000 events/sample were acquired andanalyzed using FlowJo Software.

Metaphase spreadsBone marrow cells flushed from tibias were cultured in Iscove’smodified Dulbecco’s medium (IMDM) plus 20% fetal bovineserum, murine stem cell factor (100 ng/mL), and interleukin-6(200 ng/mL) for 2 days. Cells were then exposed to 0.2 mg/mLcolcemid (Life Tech) for 4 hours and pelleted at 800 rpm for5 min. Cells were resuspended dropwise in prewarmed (37�C)75 mM KCl and incubated at 37�C for 15 min. After pelleting,cells were resuspended in a 3:1 methanol:glacial acetic acid fixa-tive. Cells were pelleted and resuspended in fixative two addi-tional times before being dropped onto slides and driedovernight. Spreads were stained with Vectashield mounting me-dium with 40,6-diamidino-2-phenylindole (DAPI, VectorLaboratories).

Histology and flow cytometryMurine tissues obtained postmortem were fixed in 10% formalin,paraffin-embedded, sectioned (5-mm sections), and stained withhematoxylin and eosin. Peripheral blood smears and bone marrowcytospins were stained with Giemsa, using the automated SiemensHematek 3000 (Fisher) system. For flow cytometry, peripheralblood cells were incubated in RBC lysis solution, and bonemarrow LDMNCS were isolated as described above. Cells werestained with either Gr-1-APC (Ly6G, clone: RB6-8C5) or B220-FITC (clone: RA3-6B2) and analyzed on a FacsCalibur machine,using live gating followed by data quantification with FlowJo soft-ware. Leukemia diagnoses were made using criteria established inthe Bethesda proposal for classification of nonlymphoid neo-plasms in mice [12], and were independently validated by a veter-inary pathologist at the Indiana University School of Medicine.

MicroscopyImages of smears, cytospins, and histologic sections were obtainedusing a Zeiss Axiolab microscope with a color camera. Metaphasespreads were imaged on a Deltavision personalDx deconvolutionmicroscope (Applied Precision). Image stacks (distance betweenz-sections: 0.2 mm) were deconvolved using Softworx andanalyzed using Imaris software suite (Bitplane).

StatisticsStatistical analysis was performed with GraphPad Prism 6 soft-ware (GraphPad Software, La Jolla, CA).

ResultsWe observed cohorts of wild-type (WT; n 5 20) andFancc�/� (n 5 18) mice for 24 months and noticed

decreased survival of Fancc�/� mice (p 5 0.01)(Fig. 1A). Five Fancc�/� mice (27.8%) died between 8and 24 months of age from leukemia or lymphoma(Fig. 1B–L). Specifically, we diagnosed AML in two mori-bund Fancc�/� mice with peripheral blasts, predominance ofGr-1þ (Ly-6G) peripheral blood LDMNCs, and myeloid in-filtrates around the liver vessels (Fig. 1C–E). One Fancc�/�

mouse developed lethal B-cell acute lymphoblastic leuke-mia (ALL), as evidenced by expansion of B220þ blaststhat replaced O90% of bone marrow and infiltrated theliver (Fig. 1F–H). Additionally, two Fancc�/� mice diedfrom metastatic abdominal T-cell lymphoma manifestedby massive mesenteric lymph node conglomerates(Fig. 1I–J) and accompanied by Cd3þ liver infiltrates(Fig. 1K–L) in the absence of bone marrow or peripheralblood abnormalities. After 24 months of observation, allsurviving Fancc�/� and WT mice were sacrificed andexamined by necropsy. Four of 13 (30.8%) 2-year-oldFancc�/� animals had hematopoietic solid tumors and/orperipheral blasts, consistent with leukemia/lymphoma. Se-rial blood counts did not reveal progressive pancytopeniain aging Fancc�/� mice, suggesting that the developmentof leukemia may not be preceded by bone marrow failurein this animal model of FA. Nine of 18 Fancc�/� micedeveloped hematopoietic malignancies by 2 years of age(including 5 animals that died prematurely from disease),compared with zero of 20 control WT mice (p 5 0.0003)(Fig. 1B). Thus, aging Fancc�/� mice are prone to hemato-poietic neoplasms, reflecting the age-dependent risk of leu-kemia in FA patients [4,10,13].

We next asked whether Fancc�/� AML can be propa-gated in WT mice via competitive stem cell transplantation.We mixed donor Fancc�/� Cd45.2þ LDMNCs isolatedfrom a moribund AML Fancc�/� mouse (Fig. 1C–E) withWT Cd45.1þ competitor LDMNCs at a 1:1 ratio and trans-planted the mixed cells into three lethally irradiated WT re-cipients. Three WT recipients of age-matched WT Cd45.2þ

LDMNCs mixed with WT Cd45.1þ LDMNCs served ascontrols (Fig. 2A). By 50 days posttransplantation, all re-cipients of Fancc�/� LDMNCs had died of AML, whereascontrol recipients of WT LDMNCs remained healthy(Fig. 2B). The diagnosis of AML was confirmed in all re-cipients by flow cytometry, peripheral blood smears(Fig. 2C), and splenomegaly (p 5 0.0216) (Fig. 2D). Pe-ripheral blood flow cytometry revealed increased Cd45.2þ

chimerism (p 5 0.0436) in recipients of leukemicFancc�/� LDMNCs compared with controls at 1 monthposttransplant (Fig. 2E, F), highlighting the malignant po-tential of leukemic Fancc�/� LDMNCs to outcompeteWT hematopoietic cells in the host marrow.

The FA signaling network maintains genomic integrityduring Fancc�/� hematopoiesis in vivo [14], and genomicinstability promotes cancer [15]. Thus, we asked whetherleukemic Fancc�/� mice exhibit increased chromosomalinstability and whether chromosomal instability precedes

Figure 1. Aging Fancc�/� mice develop hematologic malignancies. (A) Kaplan–Meier survival curve of WT (n 5 20) and Fancc�/� (n 5 18) cohorts. (B)

Table outlining incidence of leukemias and lymphomas in WT and Fancc�/� mice by 24 months of age. Statistical significance for (A) and (B) was deter-

mined using a log-rank (Mantel–Cox) test. (C) Peripheral blood smear of a moribund Fancc�/� mouse reveals leukemic blasts (arrows). (D, E) Diagnosis of

acute myeloid leukemia was confirmed with flow cytometry, which revealed (D) increased expression of the Gr1 myeloid marker in the peripheral blood

compared with WT and healthy Fancc�/� controls (E) and the presence of leukemic infiltrates in the liver. (F) Bone marrow cytospin of another moribund

Fancc�/� mouse revealed multiple blasts (arrows). (G, H) Flow cytometry indicated increased expression of the B220 B-cell marker on bone marrow blasts,

(G) and necropsy revealed leukemic infiltrates in the liver (H) consistent with B-cell ALL. (I, J) Necropsy of another Fancc�/� mouse revealed conglom-

erates of mesenteric lymph nodes. (K, L) Liver infiltrates in this mouse were Cd3þ, consistent with T-cell lymphoma.

354 D. Cerabona et al./ Experimental Hematology 2016;44:352–357

Figure 2. Transplanted Fancc�/� AML LDMNCs induce aggressive leukemia in WT recipients. (A) Schematic of competitive repopulation transplantation.

Cd45.2þ LDMNCs obtained from a leukemic Fancc�/� mouse or WT control were mixed 1:1 with Cd45.1þ WT competitor LDMNCs and transplanted into

lethally irradiated WT recipients (n 5 3 recipients per genotype). (B) Recipients of Fancc�/� LDMNCs died within 2 months of transplantation. A log-rank

(Mantel–Cox) test was used to assess significance. (C) AML in WT recipients of Fancc�/� LDMNCs. Gr1 was used as a myeloid marker for flow cytometry

(right panel). (D) Splenomegaly in moribund WT mice transplanted with Fancc�/� LDMNCs. (E, F) Leukemic Cd45.2þ Fancc�/� LDMNCs overpopulate

recipient bone marrow compared with WT Cd45.1þ competitor LDMNCs. Statistical significance was determined using an unpaired Student t test; error bars

represent SEM.

355D. Cerabona et al./ Experimental Hematology 2016;44:352–357

the onset of leukemia during Fancc�/� hematopoiesis. Wecompared karyotypes of LDMNCs isolated from leukemicFancc�/� with those of age-matched WT and healthyFancc�/� marrows. Bone marrow cells isolated fromhealthy Fancc�/� mice had a higher incidence of aneu-ploidy and an increased frequency of abnormal mitotic fig-ures compared with WT LDMNCs (Fig. 3A–D), indicatingthat Fancc�/� hematopoietic cells become chromosomallyunstable before overt leukemogenesis occurs. Similarly,FA patients develop hematopoietic chromosomal and nu-clear abnormalities prior to the onset of leukemia[14,16,17]. Leukemic Fancc�/� bone marrows weremore aneuploid (Fig. 3B) with higher mitotic indexcompared with both age-matched WT (p 5 0.001) andFancc�/� nonleukemic (p ! 0.0001) (Fig. 3D) marrows.This observation is consistent with further exacerbationof genomic instability and acquisition of bizarre karyo-

typic abnormalities reported in human FA-associatedAML [18,19].

DiscussionFancc�/� mice develop chromosomally unstable hemato-poietic malignancies as they age, recapitulating clinicaland genomic abnormalities seen in patients with Fanconianemia (Figs. 1 and 3). Interestingly, a similar incidenceof tumors has been reported in old mice deficient inanother FA core gene, Fanca, although that observationhas not reached statistical significance because of smallsample sizes [20]. Thus, late-onset carcinogenesis maybe a common phenotype of murine FA core gene knock-outs. AML arising in Fancc�/� mice can be propagatedvia hematopoietic stem cell transplant and can producerapid onset of lethal leukemia in WT transplant recipients

Figure 3. Genomic instability and abnormal mitosis in leukemic and preleukemic Fancc�/� mice. (A) Representative images of LDMNC metaphase spreads

from WT and leukemic Fancc�/� mice. (B) Increased aneuploidy in leukemic Fancc�/� LDMNCs. At least 74 spreads were counted from WT, Fancc�/�

nonleukemic, and Fancc�/� leukemic mice. Note increased chromosomal instability in nonleukemic Fancc�/� LDMNCs compared with age-matched

WT controls. Fisher’s exact test was used to determine statistical significance. (C, D) Leukemic Fancc�/� LDMNCs undergo abnormal mitosis (C) and

have a higher mitotic index (D) compared with LDMNCs from WT and Fancc�/� nonleukemic mice (n 5 3 mice/genotype; at least 500 cells were

counted per genotype). Statistical analyses were performed using c2 tests with Yates correction.

356 D. Cerabona et al./ Experimental Hematology 2016;44:352–357

(Fig. 2). As large-scale cohorts of leukemic mice areessential for preclinical drug testing, our observationsmay facilitate the development of future preclinicalmodels of FA�/� AML.

AcknowledgmentsWe thank the following funding sources: NIH K12 Pediatric Sci-entist Award, the Barth Syndrome/Bone Marrow Failure ResearchFund at Riley Children’s Foundation, the Heroes Foundation, andNIH T32 HL007910 ‘‘Basic Science Studies on Gene Therapy ofBlood Diseases’’ grant. We thank Dr. George Sandusky for his pa-thology expertise, the Indiana University In Vivo TherapeuticsCore for use of their irradiation facility, and Li Jiang for transplan-

tation assistance. We acknowledge the work of our colleagues whowe were unable to cite here because of space limitations.

Conflict of interest disclosureNo financial interests/relationships with financial interest relatingto the topic of this article have been declared.

References1. Kottemann MC, Smogorzewska A. Fanconi anaemia and the repair of

Watson and Crick DNA crosslinks. Nature. 2013;493:356–363.

2. Nalepa G, Clapp DW. Fanconi anemia and the cell cycle: New per-

spectives on aneuploidy. F1000Prime Rep. 2014;6:23.

357D. Cerabona et al./ Experimental Hematology 2016;44:352–357

3. D’Andrea AD. Susceptibility pathways in Fanconi’s anemia and breast

cancer. N Engl J Med. 2010;362:1909–1919.

4. Alter BP. Fanconi anemia and the development of leukemia. Best

Pract Res Clin Haematol. 2014;27:214–221.

5. Parmar K, D’Andrea A, Niedernhofer LJ. Mouse models of Fanconi

anemia. Mutat Res. 2009;668:133–140.

6. Chen M, Tomkins DJ, Auerbach W, et al. Inactivation of Fac in mice

produces inducible chromosomal instability and reduced fertility remi-

niscent of Fanconi anaemia. Nat Genet. 1996;12:448–451.

7. Haneline LS, Gobbett TA, Ramani R, et al. Loss of FancC function

results in decreased hematopoietic stem cell repopulating ability.

Blood. 1999;94:1–8.

8. Whitney MA, Royle G, Low MJ, et al. Germ cell defects and he-

matopoietic hypersensitivity to gamma-interferon in mice with a

targeted disruption of the Fanconi anemia C gene. Blood. 1996;

88:49–58.

9. Pulliam-Leath AC, Ciccone SL, Nalepa G, et al. Genetic disruption of

both Fancc and Fancg in mice recapitulates the hematopoietic mani-

festations of Fanconi anemia. Blood. 2010;116:2915–2920.

10. Alter BP, Giri N, Savage SA, et al. Malignancies and survival patterns

in the National Cancer Institute inherited bone marrow failure syn-

dromes cohort study. Br J Haematol. 2010;150:179–188.

11. Carreau M. Not-so-novel phenotypes in the Fanconi anemia group D2

mouse model. Blood. 2004;103:2430.

12. Kogan SC, Ward JM, Anver MR, et al. Bethesda proposals for classi-

fication of nonlymphoid hematopoietic neoplasms in mice. Blood.

2002;100:238–245.

13. Kutler DI, Singh B, Satagopan J, et al. A 20-year perspective on the

International Fanconi Anemia Registry (IFAR). Blood. 2003;101:

1249–1256.

14. Abdul-Sater Z, Cerabona D, Sierra Potchanant E, et al. FANCA safe-

guards interphase and mitosis during hematopoiesis in vivo. Exp Hem-

atol. 2015;43:1031–1046.

15. Gordon DJ, Resio B, Pellman D. Causes and consequences of aneu-

ploidy in cancer. Nat Rev Genet. 2012;13:189–203.

16. Mehta PA, Harris RE, Davies SM, et al. Numerical chromosomal

changes and risk of development of myelodysplastic syndrome: Acute

myeloid leukemia in patients with Fanconi anemia. Cancer Genet Cy-

togenet. 2010;203:180–186.

17. Barton JC, Parmley RT, Carroll AJ, et al. Preleukemia in Fanconi’s

anemia: Hematopoietic cell multinuclearity, membrane duplication,

and dysgranulogenesis. J Submicrosc Cytol. 1987;19:355–364.

18. Woo HI, Kim HJ, Lee SH, Yoo KH, Koo HH, Kim SH. Acute myeloid

leukemia with complex hypodiploidy and loss of heterozygosity of

17p in a boy with Fanconi anemia. Ann Clin Lab Sci. 2011;41:66–70.

19. Quentin S, Cuccuini W, Ceccaldi R, et al. Myelodysplasia and leuke-

mia of Fanconi anemia are associated with a specific pattern of

genomic abnormalities that includes cryptic RUNX1/AML1 lesions.

Blood. 2011;117:e161–e170.

20. Wong JC, Alon N, McKerlie C, Huang JR, Meyn MS, Buchwald M.

Targeted disruption of exons 1 to 6 of the Fanconi anemia group A

gene leads to growth retardation, strain-specific microphthalmia,

meiotic defects and primordial germ cell hypoplasia. Hum Mol Genet.

2003;12:2063–2076.